text:
Cancer Therapy: Preclinical
Identification of Novel Human CTL Epitopes and Their Agonist Epitopes of Mesothelin
Junko Yokokawa, Claudia Palena, Philip Arlen, Raffit Hassan, Mitchell Ho, Ira Pastan, Jeffrey Schlom and Kwong Y. Tsang
Junko Yokokawa
Claudia Palena
Philip Arlen
Raffit Hassan
Mitchell Ho
Ira Pastan
Jeffrey Schlom
Kwong Y. Tsang
DOI: 10.1158/1078-0432.CCR-05-0596 Published September 2005
Purpose: Mesothelin is overexpressed in many pancreatic and ovarian cancers, mesotheliomas, and other tumor types. Clinical trials are ongoing using immunotoxins to target mesothelin, and patients immunized with allogeneic pancreatic tumor cell lines have shown immune responses to previously defined mesothelin epitopes. The purpose of this study was to define novel mesothelin CTL epitopes and, more importantly, agonist epitopes that would more efficiently activate human T cells to more efficiently lyse human tumors.
Experimental Design and Results: Two novel mesothelin HLA-A2 epitopes were defined. T-cell lines generated from one of these epitopes were shown to lyse pancreatic and ovarian tumor cells. Several agonist epitopes were defined and were shown to (a) have higher affinity and avidity for HLA-A2, (b) activate mesothelin-specific T cells from normal individuals or cancer patients to a greater degree than the native epitope in terms of induction of higher levels of IFN-γ and the chemokine lymphotactin, and (c) lyse several mesothelin-expressing tumor types in a MHC-restricted manner more effectively than T cells generated using the native peptide. External beam radiation of tumor cells at nontoxic levels was shown to enhance the expression of mesothelin and other accessory molecules, resulting in a modest but statistically significant increase in tumor cell lysis by mesothelin-specific T cells.
Conclusions: The identification of novel CTL agonist epitopes supports and extends observations that mesothelin is a potential target for immunotherapy of pancreatic and ovarian cancers, as well as mesotheliomas.
Agonist peptides
Mesothelin
CTL epitopes
HLA-A2–binding peptides
Mesothelin, a glycosylphosphatidylinositol-linked glycoprotein, is a differentiation antigen of mesothelial cells. The cDNA encodes a Mr 69,000 precursor protein, which is proteolytically processed into two components. One component, corresponding to the COOH-terminal portion of the precursor, is a membrane-bound Mr 40,000 protein known as mesothelin. The other component is a Mr 30,000 protein, corresponding to the NH2-terminal–secreted protein megakaryocyte potentiating factor (1–3). In humans, mesothelin is expressed in normal mesothelium and certain epithelial cells of the tonsil, trachea, and fallopian tube (4). Mesothelin is overexpressed in a vast majority of ductal pancreatic adenocarcinomas; mesotheliomas; and adenocarcinomas of the ovary, lung, and stomach (5–14). Mesothelin has also been reported in squamous cell carcinomas of the esophagus, lung, and cervix (15).
The presence of mesothelin in many cancer cells makes it a potential target for cancer therapies. A monoclonal antibody, K1, which recognizes mesothelin, has been developed and used in preclinical studies (4, 16). Later, a high-affinity antimesothelin antibody was isolated by phage display (17) and after affinity improvement (18) was converted to a disulfide-linked Fv and fused to a 38 kDa portion of Pseudomonas exotoxin A to make immunotoxin SS1(dsFv)-PE38 (3). SS1(dsFv)-PE38 selectively inhibited experimental human lung cancer metastases in nude mice as well as showed antitumor activity against tumor cells obtained from patients with ovarian cancer and peritoneal mesotheliomas (19–21). Immunotoxins directed against mesothelin are currently in clinical trials (22, 23).
Mesothelin is also a potential target for T-cell direct immunity. Potential CTL epitopes of mesothelin specific for HLA-A2, HLA-A3, and HLA-A24 have recently been reported. CD8+ T-cell responses to these HLA-A2, HLA-A3, and HLA-A24 epitopes were detected in patients vaccinated with granulocyte macrophage-colony stimulating factor–transduced pancreatic cancer cell lines (24). These studies provided evidence of cross-priming postvaccination, which was directed against mesothelin-specific epitopes (24).
Several preclinical studies have shown that the modification of CTL epitopes to render them more immunogenic will result in enhanced antitumor responses. Recent clinical studies in melanoma and colorectal cancer have also shown that vaccination with such agonist epitopes can result in the generation of greater levels of T-cell responses in patients, which have correlated with clinical responses (25, 26). It has also recently been shown that agonist epitopes have the ability to stimulate T cells to produce more lymphotactin. Lymphotactin is a member of the C-chemokine family cloned from activated pro-T cells (27, 28). It is produced by activated CD4+ and CD8+ T cells, natural killer cells, intraepithelial γδ T cells, and mast cells (29–31). Lymphotactin is a powerful chemoattractant for CD4+ and CD8+ T cells and a moderate chemoattractant for natural killer cells (28, 30).
The study described here reports the identification and characterization of a novel mesothelin CTL epitope, and the generation of an enhancer agonist of this epitope. T-cell lines, generated from a pancreatic cancer patient with the agonist peptide, showed high levels of lysis of mesothelin-expressing tumor cells and enhanced IFN-γ and lymphotactin production when target cells were pulsed with the agonist peptide versus the native peptide. The studies reported here also show that the use of nontoxic doses of external beam radiation of tumor cells will enhance the expression of mesothelin and other accessory molecules and render tumor cells more susceptible to mesothelin-specific T-cell killing. These studies thus provide the rationale for the potential utility of the novel mesothelin agonist epitope, alone or in concert with previously defined mesothelin epitopes, in peptide- and/or vector-mediated immunotherapy protocols for the treatment of mesothelin-expressing tumors.
Cell cultures. A human pancreatic adenocarcinoma cell line CFPAC-1 (HLA-A2 positive and mesothelin positive; ref. 32), a human colon carcinoma SW1463 (HLA-A2 positive, mesothelin negative), a human ovarian adenocarcinoma cell line OVCAR-3 (HLA-A2 positive, mesothelin positive), and a human pancreatic adenocarcinoma cell line AsPC-1 (HLA-A2 negative and mesothelin positive) were purchased from American Type Culture Collection (Manassas, VA). Three HLA-A2–positive and mesothelin-positive peritoneal mesothelioma cell lines (YOU, ROB, and ORT), a human epidermoid carcinoma cell line A431 (HLA-A2 negative, mesothelin negative), and A431.H9 cell line were obtained from Dr. Raffit Hassan (National Cancer Institute, NIH, Bethesda, MD). A431 cells transfected with pMH107, pcDNA3.1(+) vector containing a full-length mesothelin cDNA are designated as A431.H9. A431.H9 is a stable transfected cell line (33). The cultures were free of Mycoplasma and were maintained in complete medium [RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen Life Technologies)]. The C1R cell line is a human plasma leukemia cell line that does not express endogenous HLA-A or HLA-B antigens (34). C1R-A2 cells are C1R cells that express a transfected genomic clone of HLA-A2.1 (35). These cells were obtained from Dr. William E. Biddison (National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD). The 174CEM-T2 cell line (T2) transport deletion mutant (36) was provided by Dr. Peter Cresswell (Yale University School of Medicine, New Haven, CT). C1R-A2 cells and T2 cells were Mycoplasma-free and were maintained in RPMI 1640 complete medium and in Iscove's modified Dulbecco's complete medium (Invitrogen Life Technologies), respectively.
Peptides. The amino acid sequence of mesothelin was scanned for matches to consensus motifs for HLA-A2–binding peptides. We used the computer algorithm from the BioInformatics and Molecule Analysis Section of NIH, developed by Parker et al. (37), which ranks potential MHC-binding peptides according to the predictive one-half-time dissociation of peptide/MHC complexes. The HLA-A2 allele was chosen because it is the most commonly expressed class I allele. Nine-mer and 10-mer peptides were synthesized if they conformed to the respective consensus motif. A panel of 10-mer mesothelin peptides (Table 1) and analogues with one–, two–, or three–amino acid substitution of P-547-556 peptide and MUC-1 peptide (38) were made by American Peptide Company (Sunnyvale, CA) with purity >90%.
Binding of human mesothelin peptides and analogue peptides to HLA-A2 molecules
Flow cytometric analysis. Dual-color flow cytometric analysis was done on T-cell lines by using the following antibody combinations: anti–CD56-FITC/anti–CD8-PE, anti–CD8-FITC/anti–CD45RA-PE, anti–CD8-FITC/anti–CD27-PE, and anti–CD8-FITC/anti–CD28 PE. Antibodies were all purchased from BD Biosciences (San Jose, CA). Staining was conducted simultaneously for 1 hour at 4°C; cells were then washed thrice with cold Ca2+- and Mg2+-free PBS, resuspended in the same buffer, and immediately analyzed using a FACScan and the CELLQuest program (BD Biosciences). Data were gathered from 10,000 live cells, stored, and used to generate results.
The procedure for analysis of dendritic cells was similar to the one described above. The following antibody combinations were used: anti–MHC-class II-FITC/anti–CD80-PE, anti–CD58-FITC/anti–CD54-PE, anti–MHC class I-FITC/anti–MHC class II-PE, and anti–IgG1-FITC/anti–IgG2a-PE (isotype controls). Antibodies to MHC class I and class II were purchased from Serotec (Oxford, United Kingdom); other antibodies were purchased from BD Biosciences. The method described by Fan et al. (19) using K1 antibody was used for the analysis of mesothelin expression on tumor cell lines. The cells were immediately analyzed using a Becton Dickinson FACScan equipped with a blue laser with an excitation of 15 mW at 488 nm. Data were gathered from 10,000 live cells, stored, and used to generate results.
Results were expressed in percentage of positive cells and mean fluorescence intensity. Mean fluorescence intensity was used to express the levels of fluorescence determined by measuring the average for all the cells in the gated fluorescence dot plot. The mean fluorescence intensity value was collected in log scale on the FACScan.
Peptide binding to HLA-A2. Binding of mesothelin peptides and the mesothelin analogues to HLA-A2 molecules was evaluated by the up-regulation of HLA-A2 expression on T2 cells as shown by flow cytometry (39).
Culture of dendritic cells from peripheral blood mononuclear cells. HLA-A2 normal donor peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood. PBMCs were separated using lymphocyte separation medium gradient (Organon Teknika, Durham, NC) as described previously (40). Dendritic cells were prepared using a modification of the procedure described by Sallusto et al. (41).
Generation of T-cell lines. Modification of the protocol described by Tsang et al. (42) was used to generate mesothelin-specific CTLs. To generate T-cell line T-1991-P-547, autologous dendritic cells were used as antigen-presenting cells (APC). Autologous nonadherent cells were then added to APCs at an effector-to-APC ratio of 10:1. Cultures were then incubated for 3 days at 37°C in a humidified atmosphere containing 5% CO2. The cultures were then supplemented with recombinant human interleukin 2 at a concentration of 20 units/mL for 7 days; the interleukin 2–containing medium was replenished every 3 days. The 3-day incubation with peptide and 7-day interleukin 2 supplement constituted one in vitro stimulation (IVS) cycle. Primary cultures were restimulated with autologous dendritic cells as described above on day 11 to begin the next IVS cycle. Autologous dendritic cells were used as APCs for three IVS cycles. Irradiated (23,000 rad) autologous EBV-transformed B cells were used as APCs after the third IVS cycle. For the restimulation with EBV-transformed B cells, peptides at a concentration of 25 μg/mL were used to pulse the autologous EBV-transformed B cells at an effector-to-APC ratio of 1:3 for restimulation. Cultures were then incubated for 3 days at 37°C in a humidified atmosphere containing 5% CO2. After removal of the peptide-containing medium, the cultures were then supplemented with recombinant human interleukin 2 at a concentration of 20 units/mL for 7 days. T-cell lines from patient 55 (T-55-P-547, T-55-P-554L/556V, T-55-P-548M/554L/556V, T-55-P-548L/554L/556V) were generated by stimulation of PBMCs with autologous dendritic cells pulsed with the P-547 or the agonist peptides using the same stimulation protocol as described above. The markers used for the analysis and identification of dendritic cells were CD11c, MHC-class II, CD80, CD54, CD58, and CD83. CD3 was also used as a negative marker.
Cytotoxic assay. Cytotoxicity assays were used as described previously (42).
Detection of cytokines. Supernatants of T cells exposed for 24 hours to peptide-pulsed autologous dendritic cells, in interleukin 2–free medium at various peptide concentrations, were screened for secretion of IFN-γ using an ELISA kit (BioSource International, Camarillo, CA) and lymphotactin using an ELISA assay (43). The results were expressed in pg/mL.
Chemotaxis assay. Chemotactic responses were examined using the method described previously (44). Briefly, blind Well Chambers (Neuroprobe, Gaithersburg, MD) with polyvinylpyrrolidone-free 5 μm pore size polycarbonate filters previously coated on one side with mouse Collagen IV (Trevigen, Gaithersburg, MD) were used. Supernatants from unstimulated T-55-P547 cells or T-55-P547 cells activated for 24 hours with 25 μg/mL of either P547 or P547-2 peptide were added to the lower chambers, and healthy donor PBMCs (7.5 × 104 cells, 75 μL in complete RPMI medium containing 1% human AB serum) were added to the upper chambers. As negative and positive controls, the lower chambers were loaded with medium alone or recombinant lymphotactin (50 ng/mL in complete RPMI medium containing 1% human AB serum), respectively. After incubation for 4 hours at 37°C, filters were removed from the chambers, fixed, and stained with Diff-Quik stain (Dade Behring, Inc., Newark, DE). Blocking assay was done using antilymphotactin antibody (R&D Systems, Inc., Minneapolis, MN). The number of cells associated with the lower side of the membranes was evaluated by direct counting of at least six ×40 objective fields for the standard samples or at least nine ×40 objective fields for the experimental samples.
Irradiation of tumor cells. Human tumor cell lines CFPAC-1, OVCAR-3, and AsPC-1 were irradiated as previously described (45, 46). Tumor cells were harvested in log-growth phase and were placed on ice and irradiated at 10 and 20 Gy by a Cesium-137 source (Gammacell-1000; AECL/Nordion, Kanata, Ontario, Canada) at a dose rate of 0.74 Gy/min. Control samples were also placed on ice but not irradiated. Irradiated and nonirradiated cells were then washed in fresh media and seeded in 75 cm2 cell culture flasks. Cells were harvested for surface marker determination after 72 hours in culture by flow cytometry.
Vaccinia virus infection of epidermoid carcinoma cells. A recombinant vaccinia vector encoding HLA-A2.1 was used for the infection of A431 and A431.H9 cells. This recombinant virus was constructed by the insertion of the HLA-A2.1 gene into the BamHI J region of the genome of the Wyeth strain of vaccinia virus as described (47). The gene is under control of the vaccinia 40k promoter (48). Target cells at a concentration of 1 × 107/mL in complete RPMI 1640 supplemented with 0.1% bovine serum albumin were incubated with equal volume of vaccinia virus (108 plaque-forming units/mL) in the same medium at 37°C for 1 hour. The cells were then adjusted to a concentration of 5 × 105/mL in complete medium and incubated for 3 hours at 37°C.
Statistical analysis. Statistical analysis of differences between means was done using a two-tailed paired t test (StatView statistical software, Abacus Concepts, Berkeley, CA).
The primary amino acid sequence of human mesothelin was analyzed for consensus motifs for novel HLA-A2–binding peptides. One 10-mer peptide and one 9-mer peptide were identified, subsequently synthesized, and investigated for binding to the HLA-A2 molecule in a T2 cell–binding assay. The amino acid sequences and the positions of these peptides are shown in Table 1. The MUC-1 peptide and a carcinoembryonic antigen HLA-A3–binding peptide (CAP-7) were used as positive and negative controls, respectively. Two of the peptides (P21-29 and P547-556) were shown to have positive binding in the T2 assay. Experiments were then conducted to compare the ability of the P21-29 and P547-556 peptides to bind HLA-A2 at various peptide concentrations. As seen in Fig. 1, the P547-556 peptide bound to HLA-A2 at higher levels than did the P21-29 peptide at concentrations of 50, 25, 12.5, and 6.25 μg/mL. The P547-556 peptide (designated as P547) was thus chosen for further study.
Binding of mesothelin peptides to HLA-A2. Peptides were analyzed for binding to T2 cell line as described in Materials and Methods. Peptides were used at concentrations of 0 to 50 μg/mL. P21 peptide (□), P547 (▪), positive control (MUC-1 peptide; +) and negative control (HLA-A3–binding peptide; ×). MFI, mean fluorescence intensity.
Studies were then initiated to determine whether mesothelin-specific T-cell lines could be established from PBMCs from an apparently healthy donor. Autologous dendritic cells were used as APCs. The specificity of the mesothelin-specific T cells generated (designated T-1991-P547) was analyzed after IVS cycle 3 (see Materials and Methods) for the ability to release IFN-γ and the chemokine lymphotactin after stimulation with autologous dendritic cells pulsed with the P547 peptide. When T-1991-P547 cells were stimulated with autologous dendritic cells pulsed with P547 peptide, the T cells produced 391 pg/mL IFN-γ and 78 pg/mL lymphotactin, whereas the use of autologous dendritic cells pulsed with control CAP1-6D peptide or dendritic cells alone did not result in any IFN-γ and lymphotactin production (i.e., <16 pg/mL). As determined by flow cytometric analysis, the T-1991-P547 cell line was 98.7% CD8 positive, 62.2% CD45RA positive, 1.1% CD28 positive, and 0.5% CD27 positive. The T-1991-P547 cell line was then analyzed for the ability to lyse mesothelin–positive and HLA-A2–positive human tumor cell lines. AsPC-1 (HLA-A2–negative and mesothelin-positive pancreatic cancer cell line) was used as a negative control. The expression of HLA-A2 and mesothelin on CFPAC-1, OVCAR-3, AsPC-1, SW1463, and C1R-A2 cell lines was analyzed by flow cytometry. As shown in Table 2, both CFPAC-1 cells and OVCAR-3 cells were lysed by the T-1991-P547 cells. No lysis was observed against AsPC-1 cells. T-1991-P547 cells lysed CFPAC-1 cells to a greater degree compared with OVCAR-3 cells. This may partly be due to the fact that a higher percentage of CFPAC-1 cells were expressing mesothelin compared with OVCAR-3 cells.
Ability of T-1991-P547 cells to lyse cancer cells expressing mesothelin
Analysis of the primary and secondary HLA-A2–binding anchor amino acid residues at positions 1, 2, 8, and 10 of the P547 peptide revealed that modification of amino acids at these positions could potentially enhance the binding ability of the peptide to the HLA-A2 molecule. For this reason, eight different analogues of P547 peptide were synthesized, as shown in Table 1, and were investigated for their binding ability to T2 cells along with the native P547 peptide. The carcinoembryonic antigen peptide CAP-7, which has previously been shown not to bind to T2 (42), was used as a negative control. As shown in Table 1, three of the eight analogues bound to HLA-A2 at higher levels than the native peptide. They were designated P547-2, P547-3, and P547-4. Experiments were then conducted to compare the ability of these three analogues to bind HLA-A2 at various concentrations. As shown in Fig. 2A, all bound to HLA-A2 at higher levels than did the native P547 peptide at all concentrations. These results indicated that these three analogues with modification in the primary anchor position 2 (position 548 of the mesothelin peptide) and position 10 (position 556 of the mesothelin peptide) as well as the secondary position 8 (position 554 of the mesothelin peptide) were potential agonists of peptide P547.
Binding of a native mesothelin peptide and its agonist peptides to HLA-A2. Peptides were analyzed for binding to T2 cell line as described in Materials and Methods. A, peptides were used at concentrations of 0 to 50 μg/mL. P547 peptide (▪), P547-2 (•), P547-3 (○), and P547-4 (▵), positive control (MUC-1 peptide; +) and negative control (HLA-A3–binding peptide; ×). Results are expressed in mean fluorescence intensity. B, comparison of the stability of HLA-A2 peptide complexes of mesothelin native and agonist peptides. T2 cells were incubated overnight with P547 (▪), P547-2 (•), P547-3 (○), and P547-4 (▵) peptides at a concentration of 25 μg/mL and then were washed free of unbound peptide and incubated with Brefeldin A to block delivery of new class I molecules to the cell surface. At the indicated times, cells were stained for the presence of surface peptide–HLA-A2 complexes. Points, relative percentage of binding compared with 100% at time 0.
We then examined the stability of the peptide-MHC complex for P547 (native), and P547-2, P547-3, and P547-4 agonist peptides. The peptides were incubated with T2 cells overnight, and the unbound peptides were washed off and the cells were then incubated with Brefeldin A to block delivery of new class I molecules to the cell surface. Cells were analyzed for the presence of peptide–HLA-A2 complexes at various time points. As shown in Fig. 2B, all three HLA-A2 agonist complexes were more stable than the P547-HLA-A2 complex over the 8-hour observation period, with P547-4 slightly more stable than the P547-2 and P547-3 complexes over the same period of time. These data indicated that both the binding to the MHC molecule and the stability of the peptide-MHC complex were greater for the P547-2, P547-3, and P547-4 agonist peptides than the native P547 peptide.
Studies were then undertaken to compare the ability of the P547-2, P547-3, and P547-4 agonist peptides to activate the T-1991-P547 cells, which were generated with the native peptide. As seen in Table 3, pulsing of APCs with P547-2 peptide led to greater levels of both IFN-γ and lymphotactin production by T-1991-P547 cells compared with P547-3, P547-4, or the native P547 peptide. Studies were then conducted to compare the ability of the agonist peptides P-547-2, P547-3, P547-4, and the native P574 peptide at various peptide concentrations to activate T-1991-P547 cells in the production of lymphotactin. As shown in Fig. 3, at concentrations of 3.13 μg/mL and higher, the pulsing of APCs with P547-2 led to the highest level of lymphotactin production by the T-1991-P547 cell line.
Production of IFN-γ and lymphotactin by T-1991-P547 cells stimulated with autologous dendritic cells pulsed with mesothelin peptide analogues
Ability of autologous dendritic cells pulsed with native and agonist mesothelin peptides to induce lymphotactin production by T cells derived from the native peptide (T-1991-P547). Peptides were used at concentrations of 0 to 25 μg/mL. Results are expressed in pg/mL.
The T-1991-P547 cell line was derived from an apparently healthy individual using the native P547 peptide. Studies were then conducted to determine whether additional T-cell lines could be generated from a patient with pancreatic cancer (patient 55) using the native or the three agonist peptides. Four mesothelin-specific T-cell lines were subsequently established and were designated T-55-P547, T-55-P547-2, T-55-P547-3, and T-55-P547-4. The T-cell lines were generated by stimulation of PBMCs from patient 55 using autologous dendritic cells. The ability of these T-cell lines to lyse mesothelin-positive and HLA-A2–positive tumor cells was then investigated. As can be seen in Fig. 4, all four T-cell lines were able to lyse the mesothelin-positive and HLA-A2–positive CFPAC-1 cells and OVCAR-3 cells but showed no lysis against AsPC-1 (mesothelin positive, HLA-A2 negative) and SW1463 (mesothelin negative, HLA-A2 positive) cells. These results show the HLA-A2 and mesothelin specificity of the lysis. Furthermore, T-55-P547-2 cells, derived using P547-2 agonist peptide, showed greater lysis of tumor cells than the T cells derived using the native P-547, or the agonist P547-3 and P547-4 peptides. This was seen at three different effector-to-target ratios.
Ability of the mesothelin-specific T-cell lines established for a pancreatic cancer patient (patient 55) to lyse tumor cells expressing native mesothelin. T-55-P547 (derived from the native peptide;
), T-55-P547-2 (derived from the agonist peptide P547-2;
), and T-55-P547-4 (derived from the agonist peptide P547-4;
). Effector-to-target ratio = 50:1, 25:1, and 12.5:1 in a 16-hour 111In release assay (see Materials and Methods). Bars, SD.
Experiments were then conducted to evaluate the ability of T-55-P547-2, T-55-P547-3, and T-55-P547-4 cells to produce lymphotactin when stimulated with the corresponding peptide. As shown in Table 4, a higher level of lymphotactin production was observed when T-55-P547-2 cells were stimulated with the agonist P547-2 peptide compared with T-55-P547-3, T-55-P547-4, and T-55-P547 when stimulated with the corresponding peptide. Lymphotactin has been reported to enhance chemotaxis responses of various immune cells. A chemotaxis assay was thus done (see Materials and Methods) to confirm the functional activity of the lymphotactin in the supernatants of the mesothelin-specific T-cell line stimulated with APCs pulsed with the P547-2 agonist peptide. T-55-P547 cells were used in this study. As seen in Fig. 5A, supernatants from T-55-P547 cells stimulated with P547-2 peptide clearly enhanced migration of PBMCs over the responses to the supernatants from the native P547 peptide and the no peptide control. Purified recombinant human lymphotactin was also used as a positive control in the assay. To ascertain that the observed chemotactin responses were due to the presence of lymphotactin, a chemotaxis assay was done with and without antilymphotactin antibody. As seen in Fig. 5B, the addition of antilymphotactin antibody inhibited the migration of PBMCs induced by the purified recombinant lymphotactin as well as the migration of PBMCs induced by the supernatants from T-55-P547 cells stimulated with APCs pulsed with the agonist peptide P547-2.
Production of lymphotactin by T-cell lines generated from a pancreatic cancer patient (patient 55) stimulated with P547 and the agonist peptides
Chemotactic responses of human PBMCs to culture supernatant of a mesothelin-specific T-cell line (T-55-P547). A, the assay was done using culture supernatant of T-55-P547 cells stimulated with autologous dendritic cells pulsed with either P547 or P547-2 peptide or without peptide for 24 hours. Recombinant lymphotactin was used at 50 ng/mL as positive control, and medium was used as negative control. Each dot in the scatter plot represents the number of cells in an independently counted field. B, the assay was done in the presence (○) or absence (•) of antilymphotactin antibody at a concentration of 30 μg/mL.
To further characterize the P547-2 peptide, an additional T-cell line was established from a colon carcinoma patient (patient 35) using P547-2 agonist peptide-pulsed autologous dendritic cells. This T-cell line was designated T-35-P547-2 and was 97.4% CD8 positive, <1% CD56 positive, 33.1% CD45RA positive, and 29.0% CD27 positive. The T-35-P547-2 cell line was shown to produce IFN-γ (100 pg/mL) and lymphotactin (459 pg/mL) when stimulated with autologous dendritic cells pulsed with P547-2 agonist peptide. As seen in Table 5, the T-35-P547-2 cell line also showed lysis of the mesothelin-positive and HLA-A2–positive CFPAC-1 cells and OVCAR-3 cells at various effector-to-target ratios but showed no lysis of the mesothelin-positive and HLA-A2–negative AsPC-1 cell line. In addition, the T-35-P547-2 cell line also lysed HLA-A2–positive, mesothelin-positive mesothelioma cell lines YOU, ROB, and ORT, as shown in Table 5. To confirm our hypothesis that tumor cells endogenously process mesothelin to present mesothelin peptide in the context of MHC for T-cell–mediated lysis, CFPAC-1 cells were used as target in a cold target inhibition assay. As shown in Table 6, the addition of peptide-pulsed unlabeled T2 cells decreased the CTL activity of T-35-P547-2 cells against labeled CFPAC-1 cells. The cytotoxic activity of T-35-P547 cells against CFPAC-1 cells was shown to be HLA-A2 restricted as indicated by the inhibition of lysis with anti–HLA-A2 antibody but not with control antibody (Table 6). To further show that target cells can endogenously process the entire mesothelin molecule in a manner so as to bind HLA-A2 molecules for presentation at the cell surface, A431 cells were transfected with the entire human mesothelin gene (see Materials and Methods) and used as target cells in a T-cell cytotoxic assay. As seen in Table 7, the mesothelin-transfected A431 cells designated as A431.H9 cells express mesothelin. A431.H9 cells were susceptible to lysis with T-35-P547-2 cells when transfected with rV-HLA-A2 recombinant expressing HLA-A2. These studies further show the endogenous process of mesothelin in A431.H9 cells and the HLA-A2–restricted nature of the mesothelin-specific lysis of T-35-P547-2 cells.
Ability of the mesothelin-specific T-cell line (T35-P547-2) to lyse human tumor cell lines expressing mesothelin
Cytotoxicity of a mesothelin-specific T-cell line (T-35-P547-2) against target cells with endogenous mesothelin expression and inhibition of lysis by anti-HLA-A2 antibody
Demonstration of HLA-A2 involvement in the ability of a mesothelin-specific T-cell line (T-35-P547-2) to lyse target cells with endogenous mesothelin expression
It has been previously shown that sublethal irradiation of tumor cells can modulate their phenotype to render them more susceptible to T-cell–mediated killing (45, 46). We investigated whether sublethal doses of radiation would alter the expression of surface markers in CFPAC-1, OVCAR-3, and AsPC-1 cell lines. Tumor cells were subjected to 0 or 10 Gy radiation, and cell surface expression of mesothelin, HLA-A2, and intercellular adhesion molecule (ICAM)-1 was analyzed by flow cytometry after 72 hours. No significant increase in cell death was observed with 10 and 20 Gy radiation. As seen in Fig. 6A, slight or no increases in the percentage of cells expressing mesothelin, HLA-A2, and ICAM-1 were detected in the CFPAC-1 cell line at 10 Gy radiation compared with no radiation. This is due to the fact that the CFPAC-1 cell line has a high level of expression of these three molecules. The mean fluorescence intensity of ICAM-1, mesothelin, and HLA-A2, however, did increase (Fig. 6A). As shown in Fig. 6B, increases in the percentage of mesothelin-expressing cells and ICAM-1–expressing cells were observed in the OVCAR-3 cell line after irradiation at 10 Gy. No significant increase in the percentage of cells expressing these three markers was observed on AsPC-1 cells after irradiation at 10 Gy. The mean fluorescence intensity of expression of mesothelin, however, did increase after irradiation (Fig. 6C). The functional significance of phenotypic changes of CFPAC-1 and OVCAR-3 cells after irradiation on the susceptibility to be lysed by mesothelin-specific T cells was investigated. The CFPAC-1 and OVCAR-3 cell lines both showed a significant (P < 0.01) increase in lysis by T-1991-P547, a mesothelin-specific T-cell line after 10 Gy of irradiation compared with the nonirradiated CFPAC-1 and OVCAR-3 cells (Fig. 7A and B). The AsPC-1 cell line, a HLA-A2–negative cell line not lysed by T-1991-P547, was used as a negative control (Fig. 7C).
Flow cytometry analysis of surface expression of mesothelin, HLA-A2 and ICAM-1 on human tumor cell lines: A, CFPAC-1 (pancreatic cancer cells); B, OVCAR-3 (ovarian cancer cells); and C, AsPC-1 (pancreatic cancer cells) without (unshaded area) and with irradiation at 10 Gy (shaded area).
Irradiation increases tumor cell sensitivity to mesothelin-specific T-cell killing. A, CFPAC-1, a pancreatic cancer cell line. B, OVCAR-3, an ovarian cancer cell line. C, AsPC-1, a pancreatic cancer cell line. Tumor cells were mock irradiated (
) and irradiated with 10 Gy (
) and cultured for 72 hours. Tumor cells were labeled with 111In and were used in a 16-hour 111In release assay (see Materials and Methods). Bars, SD.
Mesothelin has previously been shown to be a potential target for both antibody- and vaccine-mediated immunotherapy. Preclinical studies using antimesothelin immunotoxin SS1(dsFv)PE38 (SS1P) showed the antitumor activity against mesothelin-expressing tumors (20, 21). Two phase 1 clinical trials using SS1P are currently ongoing (22, 23). Mesothelin-derived HLA-A2–, HLA-A3–, and HLA-A24–restricted CTL 9-mer epitopes have been previously reported (24). These peptides were used for the detection of mesothelin-specific CD8+ T-cell immune responses in pancreatic cancer patients vaccinated with granulocyte macrophage-colony stimulating factor–transduced pancreatic cancer cells. The two HLA-A2–binding peptides used in that study were mesothelin peptides 20 to 28 (SLLFLLFSL) and 530 to 538 (VLPLTVAEV; ref. 24). The results from that clinical trial provide evidence that CD8+ T-cell responses can be generated via cross-presentation by an immunotherapy approach designed to recruit APCs to the vaccination site. In the studies reported here, we have modified the primary anchor residues of a novel mesothelin peptide to augment the binding affinity of the peptide to the MHC molecule. Eight analogues were synthesized and analyzed; one of them, designated P547-2, was shown to be superior to the native epitope in terms of affinity of binding to MHC molecules, avidity of the peptide-MHC complex, and the ability to activate CTLs in vitro.
T-cell lines derived from the native or the agonist mesothelin epitope were shown to lyse mesothelin-positive and HLA-A2–positive pancreatic cancer, ovarian cancer, and mesothelioma cell lines as well as mesothelin gene–transfected epidermoid carcinoma cells in an MHC-restricted manner. Moreover, such T-cell lines could be derived from a pancreatic cancer patient, a colon cancer patient, and a normal donor.
Preclinical and clinical studies have indicated that lymphotactin may be an important chemokine in attracting effector cells and thus enhancing immune responses. The results reported here show both enhanced synthesis of lymphotactin and enhanced biological activity of lymphotactin as a consequence of stimulation of effector T cells with the agonist mesothelin peptide.
Previous studies have shown that when murine and human tumor cells are exposed to sublethal irradiation, their phenotype can be altered to make them more susceptible to T-cell–mediated killing. This has been shown to be due to up-regulation of either tumor antigen, MHC class I, or accessory molecules such as Fas or ICAM-1, or combinations of the above. The results reported here show that nonlethal irradiation of both a pancreatic and ovarian cancer cell line led to the up-regulation of mesothelin, ICAM-1, and MHC class I, which subsequently rendered them more susceptible to lysis by a mesothelin-specific T-cell line.
The studies reported here thus extend previous observations on the suitability of mesothelin as a potential target for immunotherapy of a range of human tumors.
We thank the Stehlin Foundation for Cancer Research (Houston, TX) for providing the tumor cell lines YOU, ROB, and ORT, which were established from the ascites of patients with peritoneal mesothelioma, and Debra Weingarten for her editorial assistance in the preparation of the manuscript.
Accepted June 10, 2005.
Received March 17, 2005.
Revision received June 3, 2005.
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This weekend is the fourth round of the 2019 Formula 1 World Championship, on the streets of Baku. Here is the timetable for the major activities, including the F2 sessions.
Plans to run the proposed Miami Grand Prix around Biscayne Boulevard and Bayfront Park in the city's downtown area appear to have been scrapped in favour of a new venue adjacent to the Miami Dolphins stadium, according to the group led by the NFL team's owner.
The Netflix documentary Drive to Survive has been well received by most in Formula 1 circles despite the conspicuous absence of Ferrari and Mercedes. Will the streaming service commission a second series, and what direction could it take?
Ferrari and Mercedes have already developed their cars extensively in what has been an intriguing start to the Formula 1 season after three rounds so far.
Daniel Ricciardo believes short-term judgements on Formula 1 drivers' form are like questioning the quality of a footballer on a brief goalless streak.
Stefan Johansson, former Ferrari and McLaren F1 driver, has come up a with a template for a radically revised version of the sport he loves. Here is the second in a four-part series on what needs to change and why, this time in the nature of the competition.
Heading into the Azerbaijan Grand Prix in Baku, there are already strong battles breaking out among the Formula 1 field - giving our regular video show 'Teammate Wars' plenty to examine.
The depth and breadth of the illustrative archive of Giorgio Piola really knows no bounds, with the legendary Formula 1 illustrator having captured 50 years of the sport's technical endeavors. Join us as we continue to take a look at some of his favourite cars and this time venture into the excess and decadence of the 1980s – this time, the Brabham BT52.
Stefan Johansson, former Ferrari and McLaren Formula 1 driver, has done a lot of thinking – and writing – to come up a with a template for a radically revised version of the sport he loves. Here is the first of a four-part series on what needs to change and why.


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Ruika Sato (born 27 March 1992) is a Japanese judoka. She competed in the women's 78 kg event at the World Judo Championships in 2013, 2014 and 2017.

In 2018, she won the gold medal in the women's 78 kg event at the 2018 Asian Games held in Jakarta, Indonesia.

References

External links

Living people
1992 births
Place of birth missing (living people)
Japanese female judoka
Judoka at the 2018 Asian Games
Asian Games gold medalists for Japan
Asian Games medalists in judo
Medalists at the 2018 Asian Games
21st-century Japanese women

redpajama_set_name: RedPajamaWikipedia


text:
Four teen groupies kidnap their music idol Shea, for fun only to find themselves at the mercy of a manipulative psychopath on a deadly mission to settle old scores.
Director: Empress
Actors: David Parisian, Harry Hains, James Chilli Chillingworth, Kelly-Marie Murtha, Lisa Summers, Paul Ebejer, Peter Mazzucco, Sammi Barber, Shannon Hamm, Stephanie Herrera, Tommy James Murphy
Keywords:Groupies
Girl Missing
15 yrs after she was left in a ditch, a young woman is contacted by a millionaire from NYC who claims to be her mother. When she arrives at the…
Genre: Mystery, Thriller
Bill, a wealthy businessman, confronts his junkie daughter's drug-dealing boyfriend; in the ensuing argument, Bill kills him. Panic-stricken, he wanders the streets and eventually stops at a bar. There he…
Edge of Isolation
Stranded deep in the woods, a young married couple is taken in by an isolationist family, one that lives off the blood and plunder from lost hikers, and they want…
A rising young attorney successfully defends a man accused of murder, only to have the same type of murder then happen again. Right away the previously defended man hires the…
Genre: Crime, Drama, Romance, Thriller
American Gigolo
Julian makes a lucrative living as an escort to older women in the Los Angeles area. He begins a relationship with Michelle, a local politician's wife, without expecting any pay….
Genre: Crime, Drama, Mystery, Romance, Thriller
Run Like Hell
Every year, sisters Samantha and Maggie fly home to spend Fourth of July with their parents. But this year Samantha's new husband Dan has mapped out the perfect road trip….
Set in London's world of high class call girls, Amoureux Solitaires tells the story of London based French escort Virginie, and Rupert, a man who rescues trafficked girls.
The Keeping Hours
A supernatural love story that centers on two parents whose life together falls apart after their son dies. The man, now a successful lawyer, and the woman, now married and…
Genre: Drama, Fantasy, Horror, Romance, Thriller
A talented, but struggling actor is willing to go to any length to get a job – including "break a leg"… especially those of other actors.
Genre: Comedy, Drama, Thriller
A middle-aged man in a stagnant marriage, finds his life upended when an attractive young woman and her seemingly abusive husband move in next door.
Genre: Drama, Romance, Thriller
A Trace of Danger
A successful defense attorney finds her life in jeopardy when she returns home to defend her high school sweetheart against charges of murdering his wife.
During a rescue mission, a team of Navy Seals discover that a terrorist group have access to deadly US built Stinger missiles, and must set out to locate and destroy…
Trailer: Groupies


text:
Cenotaph takes Chelmsford feature to set up big-race ambitions
Valuable prize goes to Noseda and Moore
Sign up to Tote, get a £10 risk-free first bet.
Cenotaph ran out a clear-cut winner of the Betfred Chelmsford City Cup Handicap on Saturday night.
The Jeremy Noseda-trained six-year-old had won his last two starts over six furlongs at the Essex track, but stepped up another furlong for the £100,000 feature.
Running in the colours of Michael Tabor's wife, Doreen, and ridden by Ryan Moore, the War Front gelding travelled nicely and was full of running when quickening on the outside of runners two furlongs out.
Charles Molson kept him honest, but the 3-1 favourite was a length and a quarter too good at the line and is now three from three since joining Noseda from Aidan O'Brien.
Noseda told At The Races: "I'm delighted, when he won here last time we thought about a few things and I thought this was the race to go to. We'll have a chat with Michael in the next few days, and see where we go from there.
"I was a bit concerned coming here with the trip, I think he's a better horse at six and in the last half-furlong he was maybe treading water a bit.
"But he's got plenty of talent and I think he's probably a stakes horse in the making. He's got a great attitude and I'm thrilled with the way he's going."
He added: "I've got a little plan in mind, but I'll have a word with the boss. I've always thought he might go well on dirt, maybe America but if not, Dubai, the Golden Shaheen in Dubai."


Max Bonderchuk, 17, drops and gives them 20 at a Slavic boot camp this summer. The three-week camp's goal was to teach good habits and help teens succeed, said youth pastor and camp director Yary Golosinski. In addition to push-ups and other discipline, the camp incorporated pastoral counseling and classes on character building, resisting peer pressure and leadership. "They see how it works at boot camp and then can do it at home," Golosinski said.
Slavic Christians open a rehab program to combat increasing drug use among their children.
A youth pastor organizes a boot camp for Slavic teenagers struggling with school or rebelling against their parents' authority.
Immigrant teens flock to a new, more progressive kind of church in search of spiritual grounding without the rigid traditions that their parents' churches demand.
Such responses are beginning to take shape across the Portland area to relieve a culture clash between evangelical Christians who emigrated from the former Soviet Union to escape religious persecution and their increasingly Americanized children. The symptoms include rising school dropout rates, drug abuse, criminal activity and a deep divide between the generations.
The youths and community activists agree the defiance stems from the parents' devotion to old-world traditions, their faith's strict rules and the older generation's difficulty reconciling with American culture. Their pastors, typically resources and guides for the community, are overwhelmed and often ill-equipped to help address the youths' problems. And few other services are available to the often-isolated immigrants.
And yet, though solutions have been scarce, advocates for the estimated 100,000 Slavic immigrants living in the metro area are slowly beginning to tackle the problems. The Oregonian looks at some fresh approaches, told through the stories of three Slavic young people who, like many in their midst, are making good with their lives.
The teens prayed under a tent. They lined up in three rows, army style, then ran two laps on a gravel track through the woods. In the morning heat, they sweated through push-ups on the grass and pull-ups on chinning bars.
For three weeks in June and July, about 30 Russian-speaking boys and girls, 12 to 17 years old, attended GYM Boot Camp, a Christian program for Slavic teenagers near Camas. Established this summer by Grace Youth Ministries of Southeast Portland's Church of Grace, the camp uses discipline and pastoral counseling to change bad habits.
Southeast Portland's Church of Grace youth pastor Yary Golosinski teaches a class on character and leadership at the Slavic Christian boot camp near Camas. "The battle of whether you win or lose all starts in your minds," he tells the kids. "Maybe some guys will tell you you're not cool if you don't drink or swear or smoke. But don't give up, don't surrender to bad motives."
It was a solution to problems that Slavic teens and their parents experience, said youth pastor and camp director Yary Golosinski. Some teens who attended had only minor troubles, Golosinski said. Others were addicted to drugs or had had serious run-ins with the law. Some parents even paid their children to attend.
But the camp, where English and Russian mixed freely, was about much more than discipline. Classes centered on building character, resisting peer pressure, spiritual growth and leadership. Teens met one-on-one with a counselor and made a plan for life after camp.
"It was a good workout," 17-year-old Max Bonderchuk said. "But everyday prayer and one-on-one meetings were the best, because you had someone to talk to and give you advice."
With the Russian army as the camp leitmotif, teens followed a strict schedule of early wake-ups, workouts, classes and worship. No cell phones, no Internet. "Sergeants" led teens in paintball matches, running, team sports and trench digging. When participants misbehaved -- some brought alcohol, others swore -- they ran and did push-ups as punishment.
"We need discipline in life, and some kids here don't understand that," Golosinski said. "Once they see how it works at boot camp, they can do it at home."
The church will follow the teens and their parents for four months, making sure new habits stick.
"You don't need to have people influence you, you need to influence," Golosinski told the teens. "Don't be afraid to lose bad friends. Change in your life starts with a decision."
Lutheran Community Services Northwest: Mental health counseling, family therapy, treatment for substance abuse, parenting classes and referrals. Contact therapist Olga Parker at 503-231-7480, ext. 644, oparker@lcsnw.org, or at her private practice, Modus Vivendi, at 503-781-9065. For pastoral counseling for Slavic people, contact Il'ya Globak at 503-231-7480, ext. 626.
Portland Impact: Social and Support Services for Educational Success is a countywide case management service for Slavic students, skills groups, coaching and advocacy; contact Katie Perkins at 503-988-5961, ext. 228. Parent-child development services program provides countywide home visits and Parents As Teachers groups for Slavic population; contact Renata Wilson at 503-988-6000, or go to www.portlandimpact.org.
Immigrant and Refugee Community Organization, Young Women's Equity Project: Helps Slavic girls ages 14 to 23 prepare for careers in math, sciences, technology and engineering. Contact Margarita Vejo at 503-253-4042.
Immigrant and Refugee Community Organization, Work Force Advancement for Youth: Assists Slavic youths in making decisions about jobs through skill building, academic support and career guidance. Contact Victoria Libov at 503-234-1541, ext. 106.
Russian Youth Leadership Conference: Helps Russian-speaking, metro-area youths finish high school and go to college. Features workshops and scholarships. Next conference is March 6. Contact Russian liaison or ESL department at child's school.
Help Hotline: Run by Portland-based Church of Grace, the hot line offers free consultations for Slavic youths and families with serious problems. Call 360-903-7882.
God Will Provide Life Change Center: A residential, Christian-based, drug- and alcohol-addiction treatment center for Russian-speaking youths and adults. Contact director John Tkach at 971-404-8241, or Peter Nakhaychuk at 971-404-9939.
The camp made a difference, parents and youths said. Bonderchuk, who moved from Russia to Oregon when he was 9, had a hard time fitting in before camp. He was "in the wrong crowd," he said, and got caught shoplifting.
"My life was going down," Bonderchuk said. "It was messed up. I wanted to fix my relationship with my parents. I wanted to change. But how, I didn't know."
The camp showed him a way, he said. "I have a very good relationship with my parents right now. I also notice that I encourage other people to change when they do something that's not godly. I'm much more confident."
His mother, Tatiana Bonderchuk, agreed.
"It was awesome, when he came back to home, he was interested in his future," she said. "Now he goes to bed on time, he is interested in reading the Bible. In the camp, he revealed his emotions and realized how to spend his time effectively, how to make the right decisions."
"Our teenagers are bombarded by the Internet, by school rules and parents' rules and church rules," she said. "Sometimes they don't know who they are. But in the boot camp, they realize who they are."
Golosinski is now looking for sponsors and for other pastors' support to expand the camp next year. Some pastors, he said, don't want to acknowledge the Slavic community's problem with youths, "but if they don't help these kids now, later they will have even bigger problems."
"If our churches don't change, they will die," he said.
After a childhood of trying to fit in with American culture and a brush with crime, Alex Bolokhovskiy found his place: a less traditional Slavic church and a police career.
As a 7-year-old religious refugee who came from Russia to Vancouver with his family, Bolokhovskiy felt like an outcast. Kids taunted him because of his clothes and language. His father demanded strict discipline; his mother didn't understand him.
"In Russia, the parents' word was the law, and teachers helped parents discipline kids," said his mother, Vera. "Here, it's different. I couldn't understand the culture, couldn't grab hold of my kids to find out, because of the language. And I didn't have time, because I was working."
In high school, Bolokhovskiy said he "fell into the wrong crowd" and got involved in stealing car parts. "I wasn't thinking; I was driven by wanting to be cool," he said. But Bolokhovskiy quit the theft ring after a few months, "because it didn't feel right."
He started thinking about his life. He had been home-schooled and joined the Clark County Skills Center's criminal justice program. The career choice wasn't well received by his parents, who recalled the Soviet Union, where police terrorized people and jailed their fellow evangelical Christians.
At the time, Bolokhovskiy attended church only because his parents forced him. Rules were strict regarding behavior and dress, but no one explained why they should be followed.
"A lot of kids are forced to go to church, dress a certain way, but they would rather do what their friends do," said Bolokhovskiy, now 21. "The first chance they get, they'll do exactly the opposite of what they're told by their pastors and parents."
A Russian friend invited him to a Bible study at Word of Grace Bible Church in Battle Ground. Bolokhovskiy liked what he heard -- "more emphasis on God rather than the rules" -- and switched churches. His parents didn't approve, because some in the traditional community rebuffed Word of Grace.
The youth pastor there, Eugene Shkarovskiy, said Word of Grace isn't driven by tradition but rather by the practical application of the Bible in daily life. It's seen as "liberal" by some, he said, because it does not demand formal rules be followed.
Senior Pastor Alexey Kolomiytsev said he opened the church to help young Slavs bridge American and Russian cultures and theologies.
"We're trying to help youth think clearly and work on their habits," he said. "External issues, like appearance, are secondary to us."
The church encourages young people to preserve Russian traditions and language, but it's not a priority, Kolomiytsev said. "We fill the soul. We're not a cultural organization."
Bolokhovskiy was baptized in 2005. He's in the process of applying to work with the Clark County Sheriff's Office and often speaks to kids at church about law enforcement. Churches like Word of Grace will grow, he said, while "legalistic," rules-driven churches will slowly disappear, "because kids are leaving the strict churches in droves."
Pastor Il'ya Globak, who leads Ukrainian Bible Church, disagrees. "A church needs rules, same as a household or a company or any organization," Globak said through an interpreter. "Without rules, it's chaos. Nothing can withstand."
Alex Bolokhovskiy explains a Bible passage to Daniel Taran, (left) 12, and Eddie Altukoff, 12. The small discussion group Bolokhovskiy leads is part of a Bible study class at the Word of Grace Bible Church in Battle Ground.
But Globak said his church, which he places in the middle of the spectrum, is not "fanatic" and allows freedom. Many pastors now see change is coming, he said, while they still try to keep some rules and traditions from back home.
"Of course we try to keep our tradition and language alive," Globak said, "but the most important thing is that our kids grow up to be good, strong, exemplary people."
Life turned dark for Yelena, now 23, shortly after her family of religious refugees arrived in Portland nine years ago. She dropped out of high school in 10th grade, ran away frequently and got into trouble with the law.
She became addicted to drugs, resulting in her incarceration at the immigration detention center in Tacoma. Now her family fears she may be deported back to Moldova in Eastern Europe.
Yelena's parents faced a common problem in the Portland-Vancouver Slavic community: Few culturally specific services are available for the rising number of drug users. Only in the past three years has a single rehabilitation center opened in the metro area to offer hope to Slavic teenagers and young adults struggling with addiction.
"My daughter misunderstood the word svoboda," said Yelena's mother, Svetlana, using the Russian word for freedom. "I was in such a shock. I didn't know where to get help."
Seeking aide, Svetlana -- who asked that her family's last name not be used for fear of stigma in the community -- turned to her pastor. But he could do nothing, Svetlana said. The congregation prayed for "drug addicts."
"It's a lot, a lot of our kids," Svetlana said. "And the parents are quietly suffering, because they are going to church and afraid of the shame."
After two stints in jail and rehab, Yelena was clean for more than half a year before being arrested in October on an outstanding warrant. Her parents and siblings are U.S. citizens, but Yelena never took the examination. As a green-card holder, she is deportable for drug-related offenses.
Drug addiction among Slavic teens is on the rise, Portland area counselors and school workers say, with heroin, methamphetamine and prescription medication the drugs of choice. But because statistics aren't broken down by national origin, the trend's scope is unclear.
Olga Parker, therapist and Eastern European project coordinator at Lutheran Community Services Northwest, teaches a parenting/family dynamics class for Slavic parents. Slavic teens adapt faster to American culture than their parents do, she said, so they often play two roles -- attend church and follow the dress and behavior rules at home, and dress and behave like typical American teens when they get to school.
"While the parents attend services, the kids gather in the church parking lot smoking or being introduced to heroin," said Olga Parker, a Russian-speaking psychologist. "This is a way they may be trying to fit into American society."
A huge problem, Parker said, is the lack of beds in rehab centers for Slavic teens. Only one drug and alcohol recovery program is geared to Russian speakers in the Portland area -- the God Will Provide Life Change Center, run by Clackamas-based Fountain of Life, a Slavic evangelical congregation.
It's a free, one-year, nonmedical residential program, with six-month inpatient and six-month outpatient treatment. During the first half-year, residents work only at the center, follow strict rules, worship together and attend Bible classes. After they graduate to the adaptation program, the center helps them find a job in the community, and they begin to pay off fines or debts and learn to live without supervision.
"There is a very high demand for our program," director John Tkach said, adding that some residents are sent by their families, others by psychologists, and others by the court.
Counselors are certified through the church and live in the center. The program is working to be state certified, Tkach said, and its staff works closely with judges and probation officers.
Tkach and the congregation are looking for financial support to open a second center for girls and women. Yelena's parents hope that other programs, such as rehab and counseling, will become available at schools and in the community to help Slavic teens.
As for Yelena, who has spent more than a year at the detention center, she still doesn't know whether she will be allowed to come back to Oregon.
"I'm locked up, and I'm only in my 20s," Yelena said by phone from Tacoma. "I feel awful. I guess nobody saw that I needed help."


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Cruises to Progreso (Yucatan), Mexico
Progreso (Yucatan), Mexico
Progreso (Yucatan), Mexico Highlights
World's Longest Pier
Progreso's four-mile pier is truly one of a kind.
Featured Cruises to Progreso (Yucatan), Mexico
A small port reachable by the world's longest pier, Progreso is the perfect launching point to explore the Yucatan Peninsula. Located in the northwestern part of the Gulf of Mexico, Progreso is a tranquil town containing many beautiful white-sand beaches. You'll also have a chance to dive deep into Mexico's history by exploring ancient Mayan ruins. Its vast pier also means larger cruise ships can access Progreso.
American Discount Vacations is proud to offer the best deals possible on all cruises. Backed by our "Low Price Guarantee," and powered by our team of expert travel agents, we're sure to exceed your expectations when booking your next cruise!
Destinations for Cruises to Progreso (Yucatan), Mexico
The following cruise destinations can be visited on a cruise that includes Progreso (Yucatan), Mexico. Select a destination to view the upcoming cruises that are available:
Caribbean Cruises to Progreso (Yucatan), Mexico
Central America to Progreso (Yucatan), Mexico
Mexico Cruises to Progreso (Yucatan), Mexico
Western Caribbean Cruises to Progreso (Yucatan), Mexico
Cruise Lines with Cruises to Progreso (Yucatan), Mexico
The following cruise lines offer cruises sailing to Progreso (Yucatan), Mexico. Select a cruise line to view the upcoming cruises that visit Progreso (Yucatan), Mexico:
Filters: Show All River Specialty Mainstream
River Cruise Lines
Becoming more and more popular, a river cruise is an immerse and destination-focused vacation experience, on a small and luxurious vessel.
River cruises sail through the inland waterways of some of the world's most interesting vacation destinations (i.e. Europe, Asia, the Middle East, and North America), and provide their guests with a greater opportunity to spend time visiting more unique ports of call than a traditional cruise typically would.
Because the destination is the star of a river cruise, (typically guided) shore excursions are included at no additional charge by many River cruise lines. There are usually many interesting lectures given during the cruise, by experts on the history and culture of the region being visited.
Among the famous rivers that one may take a river cruise on are the Rhine, Mississippi, Seine, Danube, Mekong, Nile, Yangtze, Amazon and Douro.
Specialty Cruise Lines
These cruise lines offer a truly unique vacation experience, with a wide range of possible amenities and itineraries. Some are expedition cruise lines, others have sailing yachts or are coastal ships.
On these types of cruise lines, there's usually a focus on a specific destination or theme. Itineraries are usually very destination intensive, aided by the fact that they're able to reach many ports of call that larger ships can't.
Expedition cruise lines are a great fit for adventure-seeking travelers, featuring exciting destinations like the Galapagos Islands, Antarctica, and the Norwegian coast. During the cruise, there will usually be plenty of interesting lectures given by experts on the destination being visited. Often, shore excursions are included in the cost of the cruise. In fact, some of these cruise lines have all-inclusive pricing. There's a definite focus on the ship's surrounding scenery, as well as opportunities to view wildlife in their natural habitat. Small, but comfortable, ships are commonplace here, with anywhere from basic to very luxurious public spaces, cuisine, and accommodations (depending on the cruise line and ship).
Sailing Yachts, sail-assisted motor ships, offer a really unique vacation experience. Accommodations and cuisine on these intimate vessels can be anywhere from good to fantastic, depending on the cruise line. Destinations offered by these cruise lines may include the Caribbean, Europe, Panama Canal, Tahiti, and Costa Rica.
River / Specialty Cruise Line
Ships: 8 (166 - 436 Guests)
Unique U.S. Destinations
Dining, Drinks & Gratuities Included
American Queen Voyages to Progreso (Yucatan), Mexico Huge Onboard Credits!
American Queen Voyages, a consolidation of American Queen Steamboat Company and Victory Cruise Lines, offers an expanded exploration of the American heartland with river, lakes and ocean, and expedition adventures. Enjoy a relaxing and historic cruise along the beautiful rivers of the United States of America aboard one of American Queen Voyages' gorgeous paddlewheelers. While on board, you'll be transported to an era of leisurely and romantic travel, perhaps like Mark Twain did years ago. Gracious service, elegant decor, rousing entertainment and rewarding enrichment awaits you!
Learn more about American Queen Voyages
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Great Youth Program
Carnival Cruise Line to Progreso (Yucatan), Mexico Cruise Cash or Pre-Paid Gratuities!
Seuss at Sea
Carnival Cruise Line is one of the largest and most popular cruise lines in the world. Known for its festive-yet-casual atmosphere, Carnival offers a variety of on-board dining, entertainment and activity options to suit any mood or taste. Carnival's WaterWorks is a fabulous water park found onboard many of their ships. Among the many cruise destinations offered by Carnival are Alaska, the Bahamas, Canada, Caribbean, Europe and Mexico.
Learn more about Carnival Cruise Line
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Ships: 5 (1,754 - 2,500 Guests)
Disney Characters at Sea
Live Production Shows
Disney Cruise Line to Progreso (Yucatan), Mexico Special Prices!
Adult-Only Spaces
Enjoy legendary Disney service and entertainment, incomparable dining, exciting ports of call, and day after day of wonder-filled adventure. Here, specially designed areas and activities let adults, families, teens, and children play together and separately, creating magical memories. There are currently four ships in Disney's fleet, the classic Disney Magic and Disney Wonder (which are sister ships), as well as a new pair of sister ships, Disney Dream and Disney Fantasy. Disney's two new ships are their most innovative and largest yet (at a whopping 122,000-tons!).
Learn more about Disney Cruise Line
View cruise deals on Disney Cruise Line to Progreso (Yucatan), Mexico
Specialty Cruise Line
Ships: 12 (67 - 264 Guests)
Luxurious Private Yachts!
Sails to Exotic Destinations!
Ponant to Progreso (Yucatan), Mexico Huge Onboard Credits!
Butler Service in Suites!
Experience a different and magical way of cruising. Ponant prides themselves in providing modest-sized yachts that will transport you where most larger ships cannot reach. Included on board are restaurants that prepare the finest in French-inspired cuisine, as well as a well-trained and discreet staff whose goal is to provide you with the best service possible. For those who enjoy the luxurious life, some of their ships have butler service (for guests staying in select suites). All this and so much more is offered, along with all the privacy you could ask for. Come experience cruising "A la Française", the tasteful style of this company.
Learn more about Ponant
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Great Dining & Entertainment
Unique Itineraries
Princess Cruises to Progreso (Yucatan), Mexico Onboard Credit, Cash Back or Grats!
Princess Cruises has taken cruising to a whole new level, with spacious and accommodating ships packed with amenities. You'll always find the freedom to chart your own course. The Princess fleet includes a variety ships, from mid-sized to large feature-filled vessels, and features a wonderful Personal Choice Dining® program where you can choose between Traditional (assigned) and Anytime (open) dining setups to enjoy their great service and cuisine.
Learn more about Princess Cruises
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State-of-the-art Ships
Fantastic Entertainment
Royal Caribbean to Progreso (Yucatan), Mexico Onboard Credit or Pre-Paid Gratuities!
Appeals to Guests of All Ages
What type of traveler is Royal Caribbean right for? All types! Royal Caribbean is popular with families, seniors, couples, honeymooners, singles, and large groups (regardless of travel experience). Why? Because there's something for everyone on a Royal Caribbean cruise. Royal Caribbean has one of the largest fleets in the world, consisting of some of the most beautiful and modern ships in the cruising industry. Their ships are destinations within themselves! Possibilities aboard a Royal Caribbean cruise ship include Broadway-style shows, casinos that will make you think you're in Las Vegas, a variety of dining venues, great programs for children, as well as (depending on the ship), innovations like surfing simulators, sky-diving simulators, rock-climbing walls, ice-skating rinks, and much more.
Learn more about Royal Caribbean
View cruise deals on Royal Caribbean to Progreso (Yucatan), Mexico
Things to do near Progreso (Yucatan), Mexico
These are just a few of the more popular tourist attractions in Progreso, along with a brief description:
♦ Progreso Pier
Progreso is home to the world's longest pier. Stretching four miles, the Progreso Pier allows cruise ships to dock the Yucatan Coast's shallow waters. Built out of concrete with stainless-steel reinforcement, the narrow pier resembles a bridge. Take a stroll through Progreso's unique claim to fame, which is a gorgeous spot from which to watch the sunset at sea.
♦ Mayan Ruins
There are several fascinating archaeological sites featuring ancient Mayan ruins across the Yucatan Peninsula. One of the most famous is Chichen Itza, now a UNESCO World Heritage Site and one of the New 7 Wonders of the World. Explore the steep El Castillo pyramid or cool off by swimming in a cenote (a large sinkhole or natural pit) at one of Mexico's most visited places. Uxmal and Dzibilchaltun are other captivating areas to examine when traveling to Progreso.
♦ Malecon de Progreso
The lively Malecon de Progreso is teeming with shops and restaurants along a picturesque white-sand beach. Soak up the sun and get a taste of tropical paradise at one of Progreso's most beautiful beaches.
Average Monthly Weather for Progreso (Yucatan), Mexico


Water is something to celebrate as it flows through Texas' national and state parks.
Waters born from above and below, from desert downpours to crystalline springs, carve their way across the vast Texas map, shaped and colored by their geography and geology. They gradually unite and mix as tributaries become creeks, creeks become rivers and rivers become gulf. Along their transformative journeys, many will flow through tranquil places, away from the noise and trappings of civilization, each bringing beauty to the landscape and inspiring those who seek its restorative power.
Colorado Bend State Park: Spring-fed waters from Gorman Creek cascade 60 feet down multiple tiers of fern-covered travertine slopes to form Gorman Falls.
Guadalupe Mountains National Park: Shallow-water salt flats and gypsum flats are all that remain of an ancient lake, once fed by surrounding streams and by the runoff from the Guadalupe Mountains.
Caddo Lake State Park: Thick bald cypress trees and lush vegetation in tannin-colored water create a maze in the sloughs, bayous and ponds of Caddo Lake.
Big Bend National Park: Hot springs overflow from the foundation of the bathhouse built by J.O. Langford around 1909. Today, visitors still soak in the clear 105-degree springs as the Rio Grande rushes by.
Guadalupe River State Park: Framed by bald cypress trees, the Guadalupe River winds through the park in a series of deep pools and shallow rapids.
Palo Duro Canyon State Park: A red tributary of silted claystone and sandstone flows after a rainstorm on its path to the Red River.
Mustang Island State Park: Tides flow through the jetties of Mustang Island, merging waters of Corpus Christi Bay with the Gulf of Mexico.


Family owned and operated business, Beacon Plumbing can meet all of your HVAC needs in Snohomish, WA. From furnace installation to air conditioner repair, we have the tools, training and knowledge to provide you with a wide range of heating and cooling services.
As a leading new HVAC contractor, we can work on all makes and models of heating and cooling equipment. Our preventative maintenance services are designed to keep your equipment running efficiently, reliably and safely all year round.
Our commitment to quality means treating your property with utmost respect and completing jobs on time and on budget. Let help you enjoy heating and cooling comfort without disruption or unnecessary costs.
We are the most convenient and affordable HVAC repair company near you. Our team consists of licensed technicians dedicated to customer satisfaction ensuring that you receive the best possible HVAC repair services in Snohomish. Using the latest diagnostic technology, we can diagnose even the most complex issues with heating and cooling equipment.
Do not get stuck in the summer heat or winter chill; we are on call 24 hours a day, seven days a week, providing an emergency HVAC repair service in Snohomish and its surrounding areas.
Regardless of the make or model of your equipment, rely on us for an unmatched HVAC repair service performed to an excellent workmanship.
Heating and air conditioning systems are crucial to keeping your home and business comfortable and safe. When it comes to HVAC system installation, you need an experienced new HVAC contractor to help you make an informed decision about choosing new equipment.
Let us show you why we are the preferred new HVAC contractor in the Snohomish area. We look forward to serving your needs.
For same-day services of a new HVAC contractor in Snohomish, feel free to call Beacon Plumbing at (206) 452-3130.


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Idol Show Roundup: What Are You Watching?
In the fast-paced environment of the K-pop industry, staying relevant is a difficult task. Luckily, for fans and idols, variety and reality shows offer an avenue to get idols' names out there and show a different side of them. And because the entertainment industry is ever-changing, there are always new shows to check out. To help you out, we've compiled an assortment of past, present and future idols shows.
Jessica & Krystal: Featuring SNSD's Jessica and f(x)'s Krystal, the show aimed to give fans a glimpse into the personal lives and sisterhood of Jessica and Krystal. There were many emotional moments as both sisters opened up about their difficulties, and although some described the show as boring, Jessica & Krystal touched viewers with the obvious love and care the two sisters had for each other.
Hyung-don and Dae-joon's Hitmaker: In this show, MC's Hyung-don and Defconn aspired to produce an idol group made up of BTOB's Sungjae, GOT7's Jackson, and VIXX's N and Hyuk. The MC's named themselves "Brave Double Tiger" — a combination of popular producers Brave Brothers, Duble Sidekick, and Shinsadong Tiger — while the idol group was named "Big Byung," AKA "Big Bottle." In addition to the new idol group name, the members each got new nicknames in anticipation of their song "Stress Come On."
[youtube https://www.youtube.com/watch?v=_coVR9c3Fho]
BTS' American Hustle Life: Billed as a reality show in which BTS travels to L.A. to learn about hip-hop, the initial premise made many fans uneasy. Combined with the distasteful first episode in which BTS was "kidnapped" by three black men, some wrote off the show entirely.
However, those who have stuck with the show have said AHL has improved over the episodes. With the introduction of Coolio, Warren G, and Tony Jones, BTS members were tested on their hip-hop knowledge and given further challenges. If you haven't watched the show yet, be prepared for lots of secondhand embarrassment.
Idol School: Hosted by MC Jeon Hyun-moo, singer Kim Yeon-woo, and Girl's Day member Sojin, Idol School is a new variety show that puts idols to work. In order to build their own music show stages, idols must win games to receive equipment. Featuring Bestie, Halo, and members from Topp Dogg (B-joo, Hansol, A-Tom), Idol School is full of rookie K-pop idols who could use the exposure. Although unconfirmed, it seems as if future shows might feature other groups such as C-Clown, Cross Gene, and AOA.
The TaeTiSeo: Also produced by OnStyle, the network that aired Jessica & Krystal, The TaeTiSeo will focus on SNSD members Taeyeon, Tiffany, and Seohyun. The show aspires to focus on the everyday lives of the three members, including their fashion, makeup, and dining. While the premise isn't the most interesting, longtime fans of SNSD will probably find the show enjoyable.
Although we only covered a few idol shows, rest assured that these are not the only idol shows around. Many more idol shows air on YouTube channels and can be easily found with a quick search. But if your favorite idols aren't currently appearing on an idol show, you can be sure that they will sometime soon.
(OnStyle, MBC Every1, Mnet, MBC Music, Images via Lifestyler, Dispatch)
Watched/watching any of these shows? Looking forward to any other upcoming shows?
Tags: BTS, Hyuk, Jessica, Krystal, N, Seohyun, Sojin, Taeyeon, Tiffany, Yook Sung-jae
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Small firms urged to explore Korean opportunities
The Opportunity Korea programme of events around the country to help British small and medium sized businesses unlock trade opportunities in South Korea was launched today (Monday) by UK Trade & Investment.
The initiative was launched with an event at Lancaster House in London, with speakers including Business Secretary Vince Cable, Trade and Investment Minister Lord Green and International Airlines Group Chief Executive Willie Walsh.
Four more events are being held this week in Edinburgh, Birmingham, Manchester and Bristol to highlight opportunities for SMEs and offer support and advice on exporting to South Korea.
Speaking ahead of the event, Vince Cable said: "South Korea is one of the world's biggest economies and I have seen for myself the dynamic pace of growth there, and the significant potential for British business this presents.
"Investment in infrastructure projects and other major economic opportunities mean it is vital for UK companies of all sizes to maximise their own potential to export to South Korea. The skills, innovation and expertise we have in the UK can put us at the forefront of trade with this growing and vibrant economy."
Lord Green added: "South Korea is set to make the 10th largest contribution to world growth over the next five years and will become one of the most attractive export destinations globally. We want to see UK firms secure more successes this year and in the coming years. There here has never been a better time to start exporting to South Korea and I urge British companies of all sizes to explore their own potential and take advantage of the support UKTI can give them."
Willie Walsh, Chief Executive of International Airlines Group said; "I am delighted that British Airways has resumed services to Seoul six times a week. It's a booming city that offers fantastic opportunities for business and leisure with its industry, leading edge technology, excellent cuisine, fascinating history and rich culture. It is a mega city in every sense of the word — and British Airways is proud to provide the air links between the global powerhouse of South Korea to London via our home hub in award winning Terminal 5 at Heathrow."
Stephanie Hyde, head of UK regional business at Pricewaterhouse Coopers said: "With UK forecasts continuing their low trajectory, businesses already exporting to emerging economies such as China, India and Mexico, should look at increasing their activity to other regions. With strong industrial development and a growing, aspirational middle class, companies thinking of exporting to emerging economies like South Korea should be reassured that such markets can represent a significant opportunity to boost sales and increase profits.
"By having the right package of support and putting export at the heart of their strategy, businesses can succeed in reinvigorating their financial performance."
As the 12th largest economy worldwide and the fourth largest in Asia, there is great potential for UK businesses of all sizes to do business in South Korea. The introduction of the EU-South Korea Free Trade Agreement and high recognition of UK brands and products, the country is potentially one of the top overseas markets for British companies with numerous and varied opportunities for export.
Delegates attending Opportunity Korea events will be able to meet and hear from experts in doing business in South Korea including the British Ambassador Scott Wightman. In addition, representatives from British businesses already operating in South Korea will be attending to share their insights on the routes and culture of doing business there.


At Oasis Landscapes, our goal is your satisfaction. In an effort to make things easy for you, we have provided copies of some of our commonly distributed materials. Please find copies of our company brochure, our helpful plant care instruction sheet, a seasonal maintenance sheet, and limited warranty. We have also included a copy of our estimate form and employment application if you are looking to join our landscaping team.
We want your plants looking good both immediately after we install them and for many years to come. In order to help you maintain the beauty and growth of your greenery, we have created a Plant Care Instruction Guide which will give you the necessary information to keep you plants healthy and looking their best.
This guide does not cover all plants, trees, and shrubs but does give you a great overview of what to do to keep everything growing and maturing.
Special year round considerations must be followed to keep your plants happy, healthy, and looking their best. From pruning to pest control, seasonal protection against changing weather and regular fertilizing. We have constructed this helpful guide to get your plants through the entire year. Some plants, trees and shrubs require special instructions, please contact us for additional details.
Our guaranty is this. All plant material is 100% guaranteed through October 1st of the installation year. Extended warranties are available as follows: one (1) year plants, one (1) year plants and labor, two (2) year plants, and two (2) year plants and labor. Guarantee depends greatly on proper care of plant material. Replacement is one time only. Plant material not installed by Oasis Landscapes, plants subject to automated irrigation systems and accounts not paid in 30 days will void guarantee. Labor on all hard goods is guaranteed for one (1) full year.
Our extended warranty contracts are for the purpose of saving a customer charges for plant material that is subject to natural environmental conditions.
We understand that not everyone knows exactly what they are looking for before contacting us. We have created an estimate request form and it is the same form we use when meeting with customers for the first time. It helps us generate and estimate for your landscaping project.
It is not necessary to have this ready prior to our consultation, but we have found that it does sometimes provide a great start to our conversation and a place to put your thoughts and ideas together.


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Imagining and realising the exhibition - The Secret World of Butterflies - drew on the talents of prop makers, sewers, expert pinners, 3D modellers, display technicians, foley artists, curators and more.
The team worked to create several digital interactives, physical games and a giant caterpillar that is the centrepiece of the exhibition. In this feature, we look at how some of these pieces were created.
One of the most popular pieces in The Secret World of Butterflies is a large immersive digital garden where visitors can create a butterfly, colour it in and then release it into a tropical scene.
This virtual world is populated with mud-puddling butterflies, a burbling stream, butterfly-snatching birds and more.
Visualising and building this interactive experience took several months of hard graft from the digital agency Method who worked with our exhibition developers and curators.
In this video, we asked the team about some of the challenges and triumphs of creating this digital piece.
For the exhibition, we recruited a vegetable-munching, wing-flappping foley artist and a sound designer from Bespoke Post to help us create an immersive jungulous soundscape.
As you walk through the space, you will hear a fossicking pig, a frog plopping into a stream and a butterfly sucking up nectar - all of these sounds were created by one man and a wide selection of props.
In this clip, we take you into their back room to show you the ingenuity and trickery this dedicated team drew upon to create this soundscape.


Ashley Sigmon, will be here with the entire Stella & Dot collection including the NEW Summer Collection. She will be glad to help accessorize your wardrobe and/or select gifts for Spring occasions. There is jewelry for all ages! Find the perfect accessory for your Summer wardrobe and take care of any gifts you need! Stella & Dot is an awesome company that combines two of every woman's favorite things: fashion and affordability! What they sell is vintage and boutique style jewelry, along with some other accessories including bag, totes, handbags, scarves, candles, etc. Most recently: handbags, wallets, scarves, candles, and tech cases. Over half of the product line is $50 or less, and it is all beautifully made. Visa, MC, Amex, and checks accepted.
Can't wait to see you Monday, June 23rd, at 7:00 PM!
SUMMER SALE WITH DISCOUNTS UP TO 60% OFF!!!